Electrophoresis is widely used for fractionation of a variety of biomolecules, including DNA species, proteins, peptides, and derivatized amino acids One electrophoretic technique which allows rapid, high-resolution separation is capillary electrophoresis (CE) (Cohen, 1987, 1988, Compton, Kaspar). Typically, the CE employs fused silica capillary tubes whose inner diameters are between about 10-200 microns, and which can range in length between about 5-100 cm or more.
In the usual electrophoresis procedure, an electrophoresis tube, or slab, is filled with a fluid electrophoresis medium, and the fluid medium is covalently cross-linked or temperature-solidified to form a non-flowable, stabilized gel separation medium. A sample volume is drawn into or added to one end of the tube, and an electric field is placed across the tube to draw the sample through the medium. Electrophoretic separation within the matrix may be based on molecular size, in the cases of denatured protein and nucleic acid species (which have roughly the same charge density), or on a combination of size and charge, in the case of peptides and proteins.
The polymer concentration and/or degree of cross-linking of the separation medium may be varied to provide separation of species over a wide range of molecular weights and charges. For separating nucleic acid fragments greater than about 1,000 bases, for example, one preferred temperature-solidified material is agarose, where the concentration of the agarose may vary from about 0.3%, for separating fragments in the 5-60 kilobase size range, up to about 2%, for separating fragments in the 100-3,000 basepair range (Maniatis). Smaller size fragments, typically less than about 1,000 basepairs, are usually separated in cross-linked polyacrylamide. The concentration of acrylamide polymer can range from about 3.5%, for separating fragments in the 100-1,000 basepair range, up to about 20%, for achieving separation in the size range 10-100 basepairs. For separating proteins, cross-linked polyacrylamide at concentrations between about 3%-20 percent are generally suitable. In general, the smaller the molecular species to be fractionated, the higher the concentration of cross-linked polymer.
The resolution obtainable in solidified electrophoresis media of the type described above has been limited, in the case of small molecular weight species, by difficulties in forming a homogeneous, uniform polymer matrix at high polymer concentration within an electrophoresis tube, and especially within a capillary tube. In the usual method for forming a high-concentration solidified matrix in a tube, a high-concentration monomer solution, (acrylamide and bisacrylamide), is introduced in fluid form fluid into the tube. The fluid material is then polymerized, for example, by exposure to light in the presence of persulfate.
At high polymer concentrations, reaction heat gradients formed within the tube tend to produce uneven rates of reaction and heat turbulence which can lead to matrix inhomogeneities. Also, entrapped gas bubbles generated during the cross-linking reaction produce voids throughout the matrix. The non-uniformities in the matrix limit the degree of resolution which can be achieved, particularly among closely related, small molecular weight species.
Alteratively, in the case of temperature-solidifying polymers the polymer is introduced into an electrophoresis tube in a fluid form, then allowed to gel to a solid form by cooling. This approach, however, is generally unsuitable for fractionating low molecular weight species, such as small peptides and oligonucleotides, since the polymers, such as agar and agarose, which are known to have the necessary temperature-solidifying setting properties are not effective for fractionating low molecular weight species, even at high polymer concentrations.
A second limitation associated with cross-linked or temperature solidified matrices is the difficulty in recovering fractionated molecular species within the matrix, after electrophoretic separation. In the case of a preparative-scale electrophoresis tube, the solidified matrix must be carefully separated from the walls of the tube before the matrix can be removed, a procedure which is virtually impossible with small diameter tubes. Even after the matrix is removed, and the region of the matrix containing the desired molecular species is identified, the species of interest can be recovered from the matrix region only by a lengthy elution procedure, or by electrophoretic elution.
In CE, coating materials have typically been covalently attached to the walls of microcapillary tubes (Cohen, et al., 1991; Karger, et al., 1989; Alstine, et al., 1987). Most commonly polymerized matrices are introduced after covalent attachment of the coating materials (Cohen, et al., 1991; Karger, et al., 1989). Water soluble polymers have been added to cross-linked, polymerized electrophoresis medium in order to reduce brittleness, i.e., improve ease of handling, and to improve migration velocity characteristics (Ogawa, 1987; Ogawa, et al., 1990).
Grossman (U.S. patent application Ser. No. 07/731,771, now allowed) described the use of an uncharged, water-soluble polymer in a low-viscosity solution which has a mesh size useful for capillary electrophoretic separation of biopolymers.
Wiktorowicz (U.S. Pat. No. 5,015,350) described the use of non-covalent coating to adjust electroosmotic flow for the separation of biomolecules. The capillary tube is connected between anodic and cathodic electrolyte reservoirs, and an electric field is placed across the reservoirs to produce electroosmotic flow within the tube. During electroosmotic flow, a compound capable of altering the surface charge of the tube is drawn into and through the tube, and the electroosmotic flow rate within the tube is monitored. The compound is continued to be drawn into and through the tube until a desired electroosmotic flow rate in the tube, as determined from said monitoring, is achieved.
Zhu, et al., (U.S. Pat. No. 5,069,766) described the suppression of electroosmotic flow during capillary electrophoresis by the inclusion of a viscosity raising additive in one or both of the electrode chamber solutions.
Size-fractionation of protein has been performed using liquid polyacrylamide (Widhalm, et al., 1991; Takagi, et al., 1991; Bode, 1978). However, protein separations using liquid polyacrylamide in CE has attendant problems involving (i) the effect of electroosmotic flow on protein or protein-complex migration (Widhalm, et al., 1991), (ii) the use of sufficiently high polyacrylamide concentrations to attain separation of sample proteins or protein-complexes into discrete bands (Takagi, et al., 1991), and (iii) trailing of protein bands (Bode, 1978).